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WO2024073366A2 - Procédés de stabilisation d'un agent réducteur - Google Patents

Procédés de stabilisation d'un agent réducteur Download PDF

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Publication number
WO2024073366A2
WO2024073366A2 PCT/US2023/075050 US2023075050W WO2024073366A2 WO 2024073366 A2 WO2024073366 A2 WO 2024073366A2 US 2023075050 W US2023075050 W US 2023075050W WO 2024073366 A2 WO2024073366 A2 WO 2024073366A2
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Prior art keywords
reducing agent
solution
sample
preparation vessel
sample preparation
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PCT/US2023/075050
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English (en)
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WO2024073366A3 (fr
Inventor
Brittan Pasloske
Richard Crockett
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Nuclein, Llc
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Publication of WO2024073366A2 publication Critical patent/WO2024073366A2/fr
Publication of WO2024073366A3 publication Critical patent/WO2024073366A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • reducing agents are dissolved in water at a high concentration and then added to saliva infected with virus before heating to achieve the desired final concentration (Rabe 2020, Reolo 2021).
  • the saliva sample is then heated to about 90-95 °C for 5 to 10 minutes. This process of heating in the presence of TCEP is thought to inactivate ribonuclease and lyse the virus such that the viral RNA becomes competent for reverse transcription.
  • Reducing agents are frequently used as adjuvants to RNA isolation solutions in conjunction with denaturants to reduce the disulfide bonds in RNases that are rendered accessible by the denaturant.
  • RNase A, RNase 1 and RNase T1 all contain disulfide bonds (Ryle and Anfinesen, 1957; Barnard, 1969) and, therefore, are susceptible to reduction.
  • reducing agents are also used to stabilize free sulfhydryl groups in proteins and other reagents.
  • heat has been used to inactivate RNase A by mediating the breakage of disulfide bonds.
  • Zale and Klibanov (1986) performed inactivation of RNase A at 90 °C and pH 6.0 for 1 hour, which induced the following chemical changes: disulfide interchange, ⁇ - elimination of cysteine residues, and deamidation of asparagine.
  • Some reagents can be used as a catalyst increase the rate of reduction.
  • Singh and Kats (1995) demonstrated that selenol increased the reduction rate by as much as 90-fold.
  • reducing agents must be kept frozen to maintain their reducing strength, thawed when needed and then refrozen. This instability is undesirable with respect to developing room temperature stable products designed to process biological samples such that the samples are compatible with biochemical reactions such as reverse transcription.
  • TCEP and THPP are stabler than other reducing agents such as DTT in liquid form (McNulty 2015) and while they are stabler at lower pH’s, they remain susceptible to oxidation in liquid form (Han 1994).
  • the present disclosure provides methods, systems, and approaches for the preparation of biological samples comprising a reducing agent and heat.
  • the present disclosure provides methods of preparing a dried form of a reducing agent comprising: (A) obtaining a solution comprising the reducing agent, wherein the concentration of the reducing agent in the solution is greater than 20 mM; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the reducing agent.
  • the present disclosure provides methods of preparing a dried form of an organic reducing agent comprising: (A) obtaining a solution comprising an organic reducing agent; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the reducing agent.
  • the present disclosure provides methods of preparing a dried form of a tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP), or a salt thereof comprising: (A) obtaining a solution of TCEP, THPP, or a salt thereof; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the TCEP, THPP, or salt thereof.
  • TCEP tris(2-carboxyethyl)phosphine
  • THPP tris(3-hydroxypropyl)phosphine
  • the reducing agent is an organic reducing agent.
  • the reducing agent is tris(2-carboxyethyl)phosphine (TCEP), tris(3- hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT).
  • the reducing agent is a salt of a reducing agent.
  • the salt of the reducing agent is a hydrochloride salt.
  • the salt of the reducing agent is TCEP hydrochloride.
  • the methods comprise a solution with a concentration of the reducing agent of greater than 10 mM.
  • the concentration of the reducing agent is greater than 20 mM. In some embodiments, the concentration of the reducing agent is greater than 40 mM. In some embodiments, the concentration of the reducing agent is greater than 50 mM. In some embodiments, the concentration of the reducing agent is from about 20 mM to about 300 mM. In some embodiments, the concentration of the reducing agent is from about 35 mM to about 200 mM. In some embodiments, the concentration of the reducing agent is from about 50 mM to about 100 mM. In some embodiments, the solution has an acidic pH. In some embodiments, the solution has a pH of less than 6. In some embodiments, the solution has a pH of less than 5.
  • the solution has a pH from about 2 to about 6. In some embodiments, the solution has a pH from about 3 to about 5. In some embodiments, the solution has a pH of about 4.
  • the reducing agent further comprises a catalyst.
  • the catalyst is a selenol such as selenocystamine or selenocystamine dihydrochloride.
  • the methods comprise applying a fixed volume to the surface. In some embodiments, the fixed volume is applied in one spot on the surface. In other embodiments, the fixed volume is applied to two or more spots on the surface. In some embodiments, the fixed volume of reducing agent is applied in a different spot than the catalyst on the surface.
  • the fixed volume is from about 0.5 ⁇ L to about 50 ⁇ L. In some embodiments, the fixed volume is from about 1 ⁇ L to about 25 ⁇ L. In some embodiments, the fixed volume is from about 2 ⁇ L to about 10 ⁇ L.
  • the surface is a sample preparation vessel.
  • the sample preparation vessel is a plastic surface.
  • the sample preparation vessel is a glass surface.
  • the sample preparation vessel is a metal surface.
  • the methods comprise drying at a temperature greater than ⁇ 80 °C.
  • the methods comprise drying at a temperature greater than 0 °C.
  • the temperature is greater than 10 °C.
  • the temperature is from about 0 °C to about 100 °C.
  • the temperature is from about 10 °C to about 80 °C.
  • the temperature is from about 20 °C to about 60 °C. In some embodiments, the drying is carried out at a pressure greater than 0.75 atm. In some embodiments, the drying is carried out at a pressure greater than 0.9 atm. In some embodiments, the drying is carried out at a pressure greater than 1 atm.
  • the solution is an aqueous solution. In some embodiments, the solutions comprise one or more acid or base addition salts. In some embodiments, the solutions comprise an acid addition salt. In other embodiments, the solutions comprise a base addition salt. In some embodiments, the solutions comprise no other compounds other than a solvent, the reducing agent, and one or more acid or base addition salts.
  • the solutions comprise no other compounds other than a solvent and the reducing agent.
  • the present disclosure provides solid compositions of a reducing agent prepared described herein.
  • the present disclosure provides sample preparation vessels comprising: (A) a sample preparation vessel; and (B) a reducing agent; wherein the reducing agent has been affixed to an internal surface of the sample preparation vessel and wherein the reducing agent is present as a solid.
  • the reducing agent has been prepared as described herein.
  • the sample preparation vessel is a conical vial.
  • the present disclosure provides methods of preparing a raw sample comprising: (A) exposing a raw sample from a patient to a surface comprising a solid reducing agent prepared according to the methods described herein to form a reaction composition; and
  • the raw sample is a raw sample from a patient.
  • the patient is a mammal such as a human.
  • the methods comprise resuspending the reducing agent in a solution.
  • the raw sample is a biological sample such as saliva, blood, or urine.
  • the biological sample is saliva.
  • the methods further comprise analyzing the sample by PCR.
  • the PCR is RT-PCR.
  • the present disclosure provides methods of detecting an abnormal state in a patient comprising: (A) exposing a raw sample from the patient to a reducing agent, wherein the reducing agent has been prepared as described herein to obtain a lysate solution; (B) conducting PCR on the lysate solution to determine the presence of an abnormal state in the patient.
  • the raw sample is saliva.
  • the abnormal state is the presence of a disease or disorder.
  • the abnormal state is an infection of a microorganism.
  • the abnormal state is an infection of a respiratory pathogen.
  • the abnormal state is an infection of a virus.
  • the virus is a coronavirus such as the virus that causes SARS-Cov2.
  • the methods determine that a patient has an infection of SARS- Cov2.
  • the PCR is RT-PCR.
  • the PCR detects the presence of a foreign polynucleotide.
  • the present disclosure provides methods of preparing a sample comprising: (A) obtaining a sample preparation vessel comprising dried reducing agent prepared as described herein; (B) adding a raw sample to the sample preparation vessel; and (C) heating the sample preparation vessel to obtain the sample.
  • any embodiment of any of the present methods, composition, kit, and systems may consist of or consist essentially of - rather than comprise/include/contain/have - the described steps and/or features.
  • the term “consisting of” or “consisting essentially of” may be substituted for any of the open-ended linking verbs recited above, in
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof, are intended to cover a non-exclusive inclusion.
  • a process, product, article, or apparatus that comprises a list of elements is not necessarily limited only those elements but may include other elements not expressly listed or inherent to such process, process, article, or apparatus.
  • the term “or” as used herein is generally intended to mean “and/or” unless otherwise indicated. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • patient or “subject” includes mammalian organisms, such as human and non-human mammals, for example, but not limited to, rodents, mice, rats, non-human primates, companion animals such as dogs and cats as well as livestock, e.g., sheep, cow, horse, etc. Therefore, for example, although the described embodiments illustrate use of the present methods on humans, those of skill in the art would readily recognize that these methods and compositions could also be applied to veterinary medicine as well as on other animals.
  • mammalian organisms such as human and non-human mammals, for example, but not limited to, rodents, mice, rats, non-human primates, companion animals such as dogs and cats as well as livestock, e.g., sheep, cow, horse, etc. Therefore, for example, although the described embodiments illustrate use of the present methods on humans, those of skill in the art would readily recognize that these methods and compositions could also be applied to veterinary medicine as well as on other animals.
  • the present disclosure provides methods of stably manufacturing, storing or preserving reducing agents.
  • the reducing agent may be a phosphine, monothiol, dithiol, or inorganic reducing agent.
  • a non-limiting list of reducing agents include those described in, for example, Hopkins Nature, 1930, which is hereby incorporated by reference.
  • These reducing agents may be applied to one or more sample containers to allow for sample preparation.
  • the reducing agent may be affixed to a surface and then stored.
  • These containers may be for part of a kit that can further include one or more other agents and/or instructions for their use. I.
  • Reducing agents are elements or compounds that donate an electron to an oxidizer compound, hence a compound may be “reduced” (lose an electron) to create an “oxidized” state and the reaction can typically be reversed by “oxidizing” (donating an electron) a compound back into its “reduced” state.
  • reducing agents are often critical in cleaving the disulfide bonds between cysteine amino acids.
  • Commonly used thiol-based reducing reagents are ⁇ -mercaptoethanol, 2,3-bis (mercaptoethyl) pyrazine (BMMP), dithiothreitol (DTT), dithioerythritol (DTE), glutathione and cysteine.
  • ⁇ -mercaptoethanol is often included in RNA isolation solutions combined with guanidinium thiocyanate to reduce ribonuclease activity and solubilize proteins (Chomcyznski and Sacchi, 1987).
  • non-thiol based reducing agents include TRIS (2-carboxyethyl) phosphine, hydrochloride (TCEP) and TRIS (3-hydroxypropyl) phosphine (THPP). These non-thiol based reducing agents are less susceptible to oxidation and have greater stability than the thiol-based reducing agents.
  • reducing reagents are well known to those skilled in the art, and, by employing assays described herein, one of ordinary skill in the art will be able to determine which of any of these reducing agents will be of use in the present invention.
  • Presently preferred reducing agents are those comprising TCEP, THPP, DTT, ⁇ -mercaptoethanol, and cysteine.
  • the reducing agent may be a phosphine, monothiol, dithiol, or inorganic reducing agent.
  • a non-limiting list of reducing agents include those described in, for example, Hopkins Nature, 1930, which is hereby incorporated by reference. In one particular
  • the reducing agent may be TCEP (tris-(2-carboxyethyl) phosphine), THPP (tris(3-hydroxypropyl)phosphine), BMMP (2,3-bis(mercaptomethyl)pyrazine), GSH (glutathione), dithiothreitol (DTT), or dithioerythritol (DTE).
  • TCEP tris-(2-carboxyethyl) phosphine
  • THPP tris(3-hydroxypropyl)phosphine
  • BMMP 2,3-bis(mercaptomethyl)pyrazine
  • GSH glycol
  • DTT dithiothreitol
  • DTE dithioerythritol
  • the reducing agent may be TBP (tributyl phosphine), THMP (tris-(hydroxymethyl) phosphine), DHAA (dihydroasparagusic acid), BMS (bis(2-mercaptoethyl) sulfone), Meso-DTA (meso- 2,5-dimercapto-N,N,N’,N’-tetramethyladipamide), DMH (N,N’-dimethyl-N,N’- bis(mercaptoacetyl)hydrazine), DTBA ((2S)-2-amino-1,4-dimercaptobutane), DABDT (2,3- bis(mercaptomethyl)pyrazine), Sodium borohydride, Zn/H+ or Sn/H+.
  • TBP tributyl phosphine
  • THMP tris-(hydroxymethyl) phosphine
  • DHAA dihydroasparagusic acid
  • BMS
  • Ellman Assay to Measure Reducing Potential
  • a method of determining the reducing agent’s activity is needed.
  • the most common way to determine this activity or reducing potential is Ellman’s Assay (Ellman 1958).
  • Reducing activity can be accurately assayed using 5, 5'-dithiobis (2-nitrobenzoic acid) or DTNB, also known as Ellman’s reagent (Ellman 1959).
  • the reduction of DTNB mediated by a reducing agent generates a yellow color whose absorbance can be measured at 412 nm using a spectrophotometer.
  • the Ellman’s assay is used to measure the reducing potential of reducing agents such as TCEP and THPP.
  • the standard assay used to measure reducing potential was as follows. DNTB is dissolved to 0.08 mg/ml in 100 mM TRIS pH 8.0: 1 mM EDTA pH 8.0 (TE) to produce Ellman’s solution. TCEP standards were stored at -20°C to maximize their stability. They were thawed and diluted to 0.5 mM just prior to use. To assay the reducing potential of the test samples, the samples were diluted in water to generate an expected TCEP concentration of 0.5 mM. To assay each sample, 10 uL of the sample (at the expected 0.5 mM concentration) was pipetted into the well of a clear, flat-bottomed 96-well plate.
  • the reducing agent which may optionally include a catalyst, is in a shelf-stable format.
  • shelf-stable means that a reagent, formulation, or
  • v.1 manufacturing process maintains at least 50% of its original activity at a temperature above about 10°C for a period of at least 72 hours.
  • “about” means that a description may vary by ⁇ 10%.
  • the present disclosure presents methods of preparing these shelf-stable reducing agents including taking a solution with the reducing agent at an acidic pH and then drying the solution In some embodiments, the reducing agent and/or catalyst are shelf-stable.
  • the shelf-stable reducing agent and/or catalyst are additionally stable at a temperature above about 10oC, 15°C, 20°C, 25°C, 40°C, 45°C, 50°C, 60°C, 70°C, 80°C, 90°C, 95°C or higher.
  • the shelf-stable reducing agent and/or catalyst are additionally stable at a humidity above about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • the reducing agent may be in a shelf-stable solid form, optionally including a catalyst.
  • the concentration of the reducing agent for application to a surface may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM or higher.
  • the concentration of the reducing agent may be from about 1 mM to about 500 mM, from about 5 mM to about 250 mM, or from about 25 mM to about 100 mM.
  • the concentration of the reducing agent may be from about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, to about 1 M, or any range derivable therein.
  • the pH of the reducing agent and or catalyst is less than about 8, 7, 6, 5, 4, 3, 2, or 1.
  • the pH of the solution containing the reducing agent may be less than 8, 7, 6, 5, 4, 3, 2, or 1.
  • the pH of the solution containing the reducing agent may be from about 1 to about 8, from about 2 to about 6, from about 3 to about 5.
  • the pH may be from about 1, 2, 3, 4, 5, 6, 7, to about 8, or any range derivable therein.
  • the reducing agent may be applied in liquid, droplet, or vapor form to a surface. In particular embodiments, the applied reducing agent may dried on the surface.
  • the atmospheric pressure during the drying process may be greater than about 0 atm, 0.1 atm, 0.25 atm, 0.5 atm, 0.75 atm, 1 atm, or higher.
  • the pressure may be from about 0 atm to about 5 atm, from about 0.1 atm to about 2.5 atm, or
  • v.1 from about 0.25 atm to 1 atm.
  • the pressure may be from about 0.1 atm, 0.2 atm, 0.25 atm, 0.3 atm, 0.4 atm, 0.5 atm, 0.6 atm, 0.7 atm, 0.8 atm, 0.9 atm, 1.0 atm, 1.25 atm, 1.5 atm, 1.75 atm, 2.0 atm, 2.5 atm, 3.0 atm, 3.5 atm, 4.0 atm, 4.5 atm, to about 5 atm, or any range derivable therein.
  • the temperature during the drying process may be above about ⁇ 80°C, ⁇ 75°C, ⁇ 70°C, ⁇ 65°C, ⁇ 60°C, ⁇ 55°C, ⁇ 50°C, ⁇ 45°C, ⁇ 40°C, ⁇ 35°C, ⁇ 30°C, ⁇ 25°C, ⁇ 20°C, ⁇ 15°C, ⁇ 10°C, ⁇ 5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 99°C, or higher.
  • the temperature of the drying process can be from about 0°C to about 100°C, from about 5°C to about 75°C, or from about 10°C to about 50°C.
  • the temperature may be from about ⁇ 80°C, ⁇ 75°C, ⁇ 70°C, ⁇ 65°C, ⁇ 60°C, ⁇ 55°C, ⁇ 50°C, ⁇ 45°C, ⁇ 40°C, ⁇ 35°C, ⁇ 30°C, ⁇ 25°C, ⁇ 20°C, ⁇ 15°C, ⁇ 10°C, ⁇ 5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, to about 100°C, or any range derivable therein.
  • the reducing agent is dried at a temperature higher than ambient temperature and/or a pressure lower than ambient pressure to accelerate the drying process.
  • Kits for Biological Analysis The present disclosure contemplates kits that may be used to prepare biological samples.
  • the stabilized reducing agent may be coupled with one or more additives or pre- packaged in a collection tube to allow the ready processing of a raw sample.
  • the kit may further comprise one or more further active ingredients and/or instructions for the use of the reducing agents.
  • the reducing agent may be affixed to the surface of a sample container that is contained within the kit to form a usable sample. In one embodiment, the reducing agent is resuspended in water or a buffer, then mixed with the raw sample and heated.
  • the dried reducing agent is resuspended by the raw sample and heated.
  • the heated sample is subjected to PCR or RT-PCR.
  • the reducing agent is TCEP, THPP or DTT.
  • the reducing agents prepared as described herein may be used to prepare a raw sample for analysis.
  • the raw sample may be a liquid sample.
  • the liquid raw sample is saliva, urine, mucous, sputum, phlegm, blood, tears,
  • the liquid raw sample is generated by swabbing a surface, incubating the swab in a liquid for a period of time and then adding the liquid to the dried reducing agent.
  • the reducing agent is resuspended by water or a buffer and the swab immersed directly in the resuspended reducing agent.
  • the shelf-stable reducing agent and/or any other component comprised are dried on a surface with a volume less than about 500 ⁇ L, 250 ⁇ L, 100 ⁇ L, 75 ⁇ L, 50 ⁇ L, 25 ⁇ L, 15 ⁇ L, 10 ⁇ L, 9 ⁇ L, 8 ⁇ L, 7 ⁇ L, 6 ⁇ L, 5 ⁇ L, 4 ⁇ L, 3 ⁇ L, 2 ⁇ L, or 1 ⁇ L.
  • the amount of the shelf stable reducing agent or other component may be from about 1 ⁇ L to about 500 ⁇ L, from about 2.5 ⁇ L to about 100 ⁇ L, from about 5 ⁇ L to about 50 ⁇ L.
  • the amount may be from about 1 ⁇ L, 2.5 ⁇ L, 5 ⁇ L, 7.5 ⁇ L, 10 ⁇ L, 15 ⁇ L, 20 ⁇ L, 25 ⁇ L, 30 ⁇ L, 40 ⁇ L, 50 ⁇ L, 60 ⁇ L, 70 ⁇ L, 80 ⁇ L, 90 ⁇ L, 100 ⁇ L, 150 ⁇ L, 200 ⁇ L, 300 ⁇ L, 400 ⁇ L, to about 500 ⁇ L, or any range derivable therein.
  • the reducing agent may be applied in liquid, droplet, or vapor form to a surface.
  • the applied reducing agent may dried on the surface.
  • the reducing agent is applied to a surface using a spin coat method.
  • the surface is a polymer.
  • the surface is a metal or glass.
  • the surface may be an organic material.
  • the surface may be a flat or curvilinear surface.
  • the surface may comprise one or more wells or vessels for sample preparation.
  • the surface may be porous or comprise a filter material.
  • the surface may include features to facilitate mixing or turbulence of a fluid, capture of the reducing agent and/or catalyst prior to drying, or contain features to alter the probability of mixing, for example keeping the reducing agent and catalyst separate, or conversely, increasing the probability that they will combine prior to or during drying, or subsequent to drying, for example, during resuspension or dissolution.
  • the surface polymer exhibits a low surface energy that decreases the ratio of surface area to volume of the dried reducing agent and/or catalyst.
  • the polymer exhibits a high surface area that increases the ratio of surface area to volume of the dried reducing agent and/or catalyst.
  • the ratio of surface area to volume affects the drying and resuspension properties of the reducing agent and/or catalyst.
  • a low surface area to volume ratio minimizes the exposure of the reducing agent to oxidation, which may be useful to a practitioner to extend the shelf-stability of the disclosure.
  • a high surface area to volume ratio of the reducing agent and/or catalyst may increase the rate of resuspension or dissolution, or may increase the fraction that is resuspended or dissolved, which may be useful to a practitioner to decrease the amount of time or increase the efficiency of the disclosure.
  • the polymer is a polypropylene.
  • the polypropylene is a co-polymer.
  • the polypropylene is a homopolymer.
  • the polymer may be polyethylene terephthalate (PET), polystyrene, polyoxymethylene copolymer (POM) and polyvinyl styrene (PVC).
  • the reducing agent may be in a shelf-stable solid form, optionally including another component such as a catalyst.
  • the concentration of the reducing agent for application to a surface may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM or higher.
  • the concentration of the reducing agent may be from about 1 mM to about 500 mM, from about 5 mM to about 250 mM, or from about 25 mM to about 100 mM.
  • the concentration of the reducing agent may be from about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, to about 1 M, or any range derivable therein.
  • the concentration of the other components may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM or higher.
  • the concentration of the other components may be from about 1 mM to about 500 mM, from about 5 mM to about 250 mM, or from about 25 mM to about 100 mM.
  • the concentration of the other components may be from about 1 mM, 5 mM, 10 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, to about 1 M, or any range derivable therein.
  • the pH of the reducing agent and or catalyst is less than about 8, 7, 6, 5, 4, 3, 2, or 1.
  • the pH of the solution containing the reducing agent may be less than 8, 7, 6, 5, 4, 3, 2, or 1.
  • the pH of the solution containing the reducing agent may be from about 1 to about 8, from about 2 to about 6, from about 3 to about 5.
  • the pH may be from about 1, 2, 3, 4, 5, 6, 7 to about 8, or any range derivable therein.
  • a catalyst or other component may be included with the reducing agent.
  • the catalyst or other component may be mixed with the reducing agent before a drying process.
  • the catalyst or other component and reducing agent may be independently applied to the surface without mixing.
  • the reducing agent and catalyst or other component are dried on different areas of the same surface.
  • the reducing agent and catalyst or other component are dried on different areas of different surfaces.
  • the dried reducing agent and catalyst or other component are subsequently resuspended or dissolved in a liquid.
  • the reducing agent and catalyst or other component are mixed after a drying process while suspended or dissolved in a liquid.
  • one or more catalysts may be included with the reducing agent.
  • the catalyst may comprise a selenol.
  • the catalyst may be selenocystamine or selenocystamine dihydrochloride.
  • the catalyst may be a chaotrope.
  • the chaotrope may be a hydrogen bond competitor, polar destabilizer, or detergent.
  • the chaotrope may be guanidine, urea, thiourea, lithium perchlorate, lithium acetate, or an alcohol, for example, ethanol, n-butanol, isopropyl, or other alcohols which will be apparent to the person of ordinary skill.
  • the detergent may be SDS (sodium dodecyl sulfate), Triton X100, Brij, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), glycine, proline, octylglucoside, digitonin, or other detergents that will be apparent to the practitioner.
  • Example 1 Stabilization of Reducing Agents
  • TCEP Stability at Different pH’s and Temperatures TCEP solutions were prepared to 50 mM at pH 4, 7 and 9.
  • THPP Stability In a separate experiment following the same procedures, a stability study was performed for dried-down THPP (pH 4.0) on polypropylene cassettes in nitrogen. After 20 days, it was observed that the THPP maintained 100% of its reducing potential at all three incubation temperatures. At 55°C for 18 days is the equivalent of more than 6 months at 21°C. D.
  • TCEP and THPP Stability in High Humidity 8 ⁇ L of 50 mM TCEP (pH 4.0) and 50 mM THPP were pipetted onto polypropylene cassettes. They were incubated at 40oC until the spots were dry which was about 3.5 h. The cassettes were put into mylar bags with a damp cloth and a humidity logger and then sealed.
  • the saliva was spiked with gamma-irradiated SARS-CoV-2 (from BEI) to a concentration of 1 ⁇ 10 3 copies per mL of saliva. Quantities of 150 ⁇ L of the spiked saliva were pipetted into the TCEP tubes. The tubes were incubated 90°C for 7 minutes and then removed from the heat to room temperature.

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Abstract

La présente invention concerne des procédés de stabilisation d'agents réducteurs destinés à être utilisés dans des procédés biologiques. Ces agents réducteurs comprennent des agents réducteurs non à base de thiol qui sont préparés à un pH acide et/ou à une concentration élevée. Ces agents réducteurs peuvent être utilisés dans des procédés de diagnostic qui analysent des acides nucléiques tels que la PCR.
PCT/US2023/075050 2022-09-26 2023-09-25 Procédés de stabilisation d'un agent réducteur WO2024073366A2 (fr)

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